Characterization of n-glycan mixtures by nuclear magnetic resonance

ABSTRACT

The present disclosure provides nuclear magnetic resonance (NMR) methods for characterizing mixtures of N-linked glycans. Without limitation, methods of the present disclosure may be useful in characterizing monosaccharide composition, branching, fucosylation, sulfation, phosphorylation, sialylation linkages, presence of impurities and/or efficiency of a labeling procedure (e.g., labeling with a fluorophore such as 2-AB). In certain embodiments, the methods can be used quantitatively. In certain embodiments, the methods can be combined with enzymatic digestion to further characterize glycan mixtures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/595,940, filed Oct. 14, 2009, which claims the benefit under 35U.S.C. §371 of International Application Number PCT/US08/60328, filedApr. 15, 2008, which claims priority under 35 U.S.C. §119(e) to U.S.provisional application, Ser. No. 60/923,686, filed Apr. 16, 2007, theentire contents of each of which are incorporated herein by reference.

BACKGROUND

Many drugs in use today are “small molecule drugs.” These drugs exist assimple chemical structures that are synthetically derived. The activeingredient generally exists as a homogenous product. These smallmolecule drugs and preparations thereof, can be chemically characterizedusing a variety of analytical tools and are generally readilymanufactured through comparatively simple chemical synthesis.

A typical glycoprotein product differs substantially in terms ofcomplexity from a typical small molecule drug. In particular, the sugarstructures attached to the amino acid backbone of a glycoprotein canvary structurally in many ways including, sequence, branching, sugarcontent, and heterogeneity. Thus, glycoprotein products can be complexheterogeneous mixtures of many structurally diverse molecules whichthemselves have complex glycan structures. N-linked glycans are animportant class of branched sugars found in glycoproteins which have aconserved core structure with variations in branching and substitutionsof the sugar residues. Glycosylation adds not only to the moleculesstructural complexity but affects or conditions many of a glycoprotein'sbiological and clinical attributes.

To date, the creation of glycoprotein drugs having defined properties,whether an attempt to produce a generic version of an existing drug orto produce a second generation or other glycoprotein having improved ordesirable properties has been challenging due to the difficulty insynthesizing and characterizing these complex chemical structures andmixtures that contain them.

The situation with regard to the production of generic products isindicative of the problems faced in making glycoprotein drugs havingdefined properties. While abbreviated regulatory procedures have beenimplemented for generic versions of small molecule drug products, manyin the biotechnology and pharmaceutical industry have taken the viewthat the complexity of glycoprotein drug products makes them unsuitablefor similar approaches.

There is therefore a need in the art for methods for characterizingglycoproteins. In particular, there is a need for analytical methodsthat are capable of characterizing complex mixtures of glycans, e.g.,mixtures obtained by cleaving a plurality of N-glycans from aglycoprotein preparation.

SUMMARY

The present disclosure provides nuclear magnetic resonance (NMR) methodsfor characterizing mixtures of N-linked glycans. In general, NMR hasdifficulty analyzing mixtures of different molecules because it is verydifficult, often impossible, to tell which signals in an NMR spectrumcome from which molecules. This is particularly true when the mixtureincludes complex molecules and especially if they share common chemicalstructures, e.g., a mixture of N-glycans. Indeed, NMR spectra of glycansare highly complex and heavily overlapping with most ¹H signalsoccurring within the chemical shift range of 3.5-5.5 ppm. Unexpectedly,we have been able to show that subtle differences in glycan structurescan give rise to signal shifts which can be resolved and thereforequantified by two dimensional (or in some cases one dimensional) NMRexperiments. The present disclosure therefore solves the aforementionedchallenges in part by identifying NMR signals that can be resolved inspectra of glycan mixtures and that are diagnostic of particular glycanstructural features. Without limitation, methods of the presentdisclosure may be useful in characterizing monosaccharide composition,branching, fucosylation, sulfation, phosphorylation, sialylationlinkages, presence of impurities and/or efficiency of a labelingprocedure (e.g., labeling with a fluorophore such as 2-AB). In certainembodiments, the methods can be used quantitatively. In certainembodiments, the methods can be combined with enzymatic digestion tofurther characterize glycan mixtures.

Definitions

Approximately, About, Ca.: As used herein, the terms “approximately”,“about” or “ca.,” as applied to one or more values of interest, refer toa value that is similar to a stated reference value. In certainembodiments, the terms “approximately”, “about” or “ca.,” refer to arange of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of thestated reference value.

Biological sample: The term “biological sample”, as used herein, refersto any solid or fluid sample obtained from, excreted by or secreted byany living cell or organism, including, but not limited to, tissueculture, bioreactors, human or animal tissue, plants, fruits,vegetables, single-celled microorganisms (such as bacteria and yeasts)and multicellular organisms. For example, a biological sample can be abiological fluid obtained from, e.g., blood, plasma, serum, urine, bile,seminal fluid, cerebrospinal fluid, aqueous or vitreous humor, or anybodily secretion, a transudate, an exudate (e.g., fluid obtained from anabscess or any other site of infection or inflammation), or fluidobtained from a joint (e.g., a normal joint or a joint affected bydisease such as a rheumatoid arthritis, osteoarthritis, gout or septicarthritis). A biological sample can also be, e.g., a sample obtainedfrom any organ or tissue (including a biopsy or autopsy specimen), cancomprise cells (whether primary cells or cultured cells), mediumconditioned by any cell, tissue or organ, tissue culture.

Cell-surface glycoprotein: As used herein, the term “cell-surfaceglycoprotein” refers to a glycoprotein, at least a portion of which ispresent on the exterior surface of a cell. In some embodiments, acell-surface glycoprotein is a protein that is positioned on thecell-surface such that at least one of the glycan structures is presenton the exterior surface of the cell.

Cell-surface glycan: A “cell-surface glycan” is a glycan that is presenton the exterior surface of a cell. In many embodiments, a cell-surfaceglycan is covalently linked to a polypeptide as part of a cell-surfaceglycoprotein. A cell-surface glycan can also be linked to a cellmembrane lipid.

Glycan: As is known in the art and used herein “glycans” are sugars.Glycans can be monomers or polymers of sugar residues, but typicallycontain at least three sugars, and can be linear or branched. A glycanmay include natural sugar residues (e.g., glucose, N-acetylglucosamine,N-acetyl neuraminic acid, galactose, mannose, fucose, hexose, arabinose,ribose, xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose,2′-deoxyribose, phosphomannose, 6′-sulfo N-acetylglucosamine, etc). Theterm “glycan” includes homo and heteropolymers of sugar residues. Theterm “glycan” also encompasses a glycan component of a glycoconjugate(e.g., of a glycoprotein, glycolipid, proteoglycan, etc.). The term alsoencompasses free glycans, including glycans that have been cleaved orotherwise released from a glycoconjugate.

Glycan preparation: The term “glycan preparation” as used herein refersto a set of glycans obtained according to a particular productionmethod. In some embodiments, glycan preparation refers to a set ofglycans obtained from a glycoprotein preparation (see definition ofglycoprotein preparation below).

Glycoconjugate: The term “glycoconjugate”, as used herein, encompassesall molecules in which at least one sugar moiety is covalently linked toat least one other moiety. The term specifically encompasses allbiomolecules with covalently attached sugar moieties, including forexample N-linked glycoproteins, O-linked glycoproteins, glycolipids,proteoglycans, etc.

Glycoform: The term “glycoform”, is used herein to refer to a particularform of a glycoconjugate. That is, when the same backbone moiety (e.g.,polypeptide, lipid, etc) that is part of a glycoconjugate has thepotential to be linked to different glycans or sets of glycans, theneach different version of the glycoconjugate (i.e., where the backboneis linked to a particular set of glycans) is referred to as a“glycoform”.

Glycolipid: The term “glycolipid” as used herein refers to a lipid thatcontains one or more covalently linked sugar moieties (i.e., glycans).The sugar moiety(ies) may be in the form of monosaccharides,disaccharides, oligosaccharides, and/or polysaccharides. The sugarmoiety(ies) may comprise a single unbranched chain of sugar residues ormay be comprised of one or more branched chains. In certain embodiments,sugar moieties may include sulfate and/or phosphate groups. In certainembodiments, glycoproteins contain O-linked sugar moieties; in certainembodiments, glycoproteins contain N-linked sugar moieties.

Glycoprotein: As used herein, the term “glycoprotein” refers to aprotein that contains a peptide backbone covalently linked to one ormore sugar moieties (i.e., glycans). As is understood by those skilledin the art, the peptide backbone typically comprises a linear chain ofamino acid residues. In certain embodiments, the peptide backbone spansthe cell membrane, such that it comprises a transmembrane portion and anextracellular portion. In certain embodiments, a peptide backbone of aglycoprotein that spans the cell membrane comprises an intracellularportion, a transmembrane portion, and an extracellular portion. Incertain embodiments, methods of the present disclosure comprise cleavinga cell surface glycoprotein with a protease to liberate theextracellular portion of the glycoprotein, or a portion thereof, whereinsuch exposure does not substantially rupture the cell membrane. Thesugar moiety(ies) may be in the form of monosaccharides, disaccharides,oligosaccharides, and/or polysaccharides. The sugar moiety(ies) maycomprise a single unbranched chain of sugar residues or may comprise oneor more branched chains. In certain embodiments, sugar moieties mayinclude sulfate and/or phosphate groups. Alternatively or additionally,sugar moieties may include acetyl, glycolyl, propyl or other alkylmodifications. In certain embodiments, glycoproteins contain O-linkedsugar moieties; in certain embodiments, glycoproteins contain N-linkedsugar moieties. In certain embodiments, methods disclosed hereincomprise a step of analyzing any or all of cell surface glycoproteins,liberated fragments (e.g., glycopeptides) of cell surface glycoproteins,cell surface glycans attached to cell surface glycoproteins, peptidebackbones of cell surface glycoproteins, fragments of suchglycoproteins, glycans and/or peptide backbones, and combinationsthereof.

Glycosidase: The term “glycosidase” as used herein refers to an agentthat cleaves a covalent bond between sequential sugars in a glycan orbetween the sugar and the backbone moiety (e.g. between sugar andpeptide backbone of glycoprotein). In some embodiments, a glycosidase isan enzyme. In certain embodiments, a glycosidase is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a glycosidase is a chemical cleavage agent.

Glycosylation pattern: As used herein, the term “glycosylation pattern”refers to the set of glycan structures present on a particular sample.For example, a particular glycoconjugate (e.g., glycoprotein) or set ofglycoconjugates (e.g., set of glycoproteins) will have a glycosylationpattern. In some embodiments, reference is made to the glycosylationpattern of cell-surface glycans. A glycosylation pattern can becharacterized by, for example, the identities of glycans, amounts(absolute or relative) of individual glycans or glycans of particulartypes, degree of occupancy of glycosylation sites, etc., or combinationsof such parameters.

Glycoprotein preparation: A “glycoprotein preparation”, as that term isused herein, refers to a set of individual glycoprotein molecules, eachof which comprises a polypeptide having a particular amino acid sequence(which amino acid sequence includes at least one glycosylation site) andat least one glycan covalently attached to the at least oneglycosylation site. Individual molecules of a particular glycoproteinwithin a glycoprotein preparation typically have identical amino acidsequences but may differ in the occupancy of the at least oneglycosylation sites and/or in the identity of the glycans linked to theat least one glycosylation sites. That is, a glycoprotein preparationmay contain only a single glycoform of a particular glycoprotein, butmore typically contains a plurality of glycoforms. Differentpreparations of the same glycoprotein may differ in the identity ofglycoforms present (e.g., a glycoform that is present in one preparationmay be absent from another) and/or in the relative amounts of differentglycoforms.

N-glycan: The term “N-glycan”, as used herein, refers to a polymer ofsugars that has been released from a glyconjugate but was formerlylinked to the glycoconjugate via a nitrogen linkage (see definition ofN-linked glycan below).

N-linked glycans: N-linked glycans are glycans that are linked to aglycoconjugate via a nitrogen linkage. A diverse assortment of N-linkedglycans exists, but is typically based on the common corepentasaccharide (Man)₃(GlcNAc)(GlcNAc).

O-glycan: The term “O-glycan”, as used herein, refers to a polymer ofsugars that has been released from a glycoconjugate but was formerlylinked to the glycoconjugate via an oxygen linkage (see definition ofO-linked glycan below).

O-linked glycans: O-linked glycans are glycans that are linked to aglycoconjugate via an oxygen linkage. O-linked glycans are typicallyattached to glycoproteins via N-acetyl-D-galactosamine (GalNAc) or viaN-acetyl-D-glucosamine (GlcNAc) to the hydroxyl group of L-serine (Ser)or L-threonine (Thr). Some O-linked glycans also have modifications suchas acetylation and sulfation. In some instances O-linked glycans areattached to glycoproteins via fucose or mannose to the hydroxyl group ofL-serine (Ser) or L-threonine (Thr).

Phosphorylation: As used herein, the term “phosphorylation” refers tothe process of covalently adding one or more phosphate groups to amolecule (e.g., to a glycan).

Protease: The term “protease” as used herein refers to an agent thatcleaves a peptide bond between sequential amino acids in a polypeptidechain. In some embodiments, a protease is an enzyme (i.e., a proteolyticenzyme). In certain embodiments, a protease is a protein (e.g., aprotein enzyme) comprising one or more polypeptide chains. In certainembodiments, a protease is a chemical cleavage agent.

Protein: In general, a “protein” is a polypeptide (i.e., a string of atleast two amino acids linked to one another by peptide bonds). Proteinsmay include moieties other than amino acids (e.g., may be glycoproteins)and/or may be otherwise processed or modified. Those of ordinary skillin the art will appreciate that a “protein” can be a completepolypeptide chain as produced by a cell (with or without a signalsequence), or can be a functional portion thereof. Those of ordinaryskill will further appreciate that a protein can sometimes include morethan one polypeptide chain, for example linked by one or more disulfidebonds or associated by other means.

Sialic acid: The term “sialic acid”, as used herein, is a generic termfor the N- or O-substituted derivatives of neuraminic acid, anine-carbon monosaccharide. The amino group of neuraminic acid typicallybears either an acetyl or a glycolyl group in a sialic acid. Thehydroxyl substituents present on the sialic acid may be modified byacetylation, methylation, sulfation, and phosphorylation. Thepredominant sialic acid is N-acetylneuraminic acid (Neu5Ac). Sialicacids impart a negative charge to glycans, because the carboxyl grouptends to dissociate a proton at physiological pH. Exemplary deprotonatedsialic acids are as follows:

Signal integral: As used herein, the terms “signal integral” refer tothe magnitude of a particular signal (including cross-peaks) within anNMR spectrum. In one embodiment, the signal integral is obtained bymeasuring the signal area (for peaks in a one dimensional spectrum) orsignal volume (for cross-peaks in a multi-dimensional spectrum). In oneembodiment, the signal integral is obtained by measuring the signalintensity.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the structure of the common core pentasaccharide(Man)₃(GlcNAc)(GlcNAc) of N-glycans.

FIG. 2 shows the structure of an exemplary tetrasialo tetraantennaryfucosylated N-glycan. The numbering scheme used to identify the residuesthroughout the text is shown.

FIG. 3 shows the structures of the standard N-glycans (a) A1F, (b) NA3and (c) NA4.

FIGS. 4A-4U shows the structures of exemplary N-glycans.

FIG. 5 shows the 1D ¹H spectra of (a) A1F, (b) NA3, (c) NA4 and (d) amixture of N-glycans. The spectra were acquired on a 600 MHz BrukerAvance spectrometer with 5 mm cryoprobe at 27° C. with presaturation ofthe water resonance. Each spectrum is the average of 16 to 256 scans.The recycle delay was 14s.

FIG. 6 shows the anomeric region of the 1D ¹H spectrum of a mixture ofN-glycans. Potential oligomannose structures are indicated with anasterisk (*).

FIG. 7 shows part of the 1D ¹H spectrum of a mixture of N-glycansacquired on a 600 MHz Bruker Avance spectrometer with 5 mm cryoprobe, at25° C., with presaturation of the water resonance.

FIG. 8 shows the 2D ¹H-¹H TOCSY spectrum of A1F. The spectrum wasacquired on a 600 MHz Bruker Avance spectrometer with 5 mm cryoprobe at27° C. using 120 ms MLEV-17 mixing. A total of 4 points were averagedfor each of 4096×256 hypercomplex points. The recycle delay was 1.4 s.

FIG. 9 shows the overlaid 2D ¹H-¹H TOCSY spectra of the anomeric regionsof A1F (black), NA3 (red), and NA4 (green). The H1/H2 cross-peakposition of Man4′ is diagnostic branching, with the only bisubstitutedspecies, NA4, showing a distinct shift from the others. The H2/H3cross-peak position of Man4 is diagnostic of branching, with the onlymonosubstituted species, A1F, showing a distinct shift from the others.

FIG. 10 shows the 2D ¹H-¹³C HSQC spectrum of A1F. The spectrum wasacquired on a 600 MHz Bruker Avance spectrometer with 5 mm cryoprobe at27° C. using a sensitivity-enhanced gradient HSQC pulse sequence. Atotal of 16 points were averaged for each of 1024×256 hyper complexpoints. The recycle delay was 1.1 s.

FIG. 11 shows the overlaid 2D ¹H-¹³C HSQC spectra of the anomericregions of A1F (black), NA3 (red), and NA4 (green).

FIG. 12 shows the 2D ¹H-¹³C HSQC spectrum of an N-glycan pool, recordedat 27° C., in D₂O, with a 600 MHz Bruker Avance spectrometer with 5 mmcryoprobe. The numbering scheme used to identify the residues isindicated in FIG. 1. GlcNAc_(ext) stands for N-acetylglucosamine inlactosamine extension; Gal_(ext) indicates galactose in lactosamineextension. An asterisk (*) indicates signals assigned to oligomannosestructures.

FIG. 13 shows the anomeric region of the 1D ¹H spectra of unlabeled and2-AB labeled N-glycan pools. Spectra were recorded at 600 MHz, 25° C.,in D₂O.

FIG. 14 shows the 2D ¹H-¹³C HSQC spectrum of a 2AB-labeled N-glycanpool, recorded at 27° C., in D₂O, with a 600 MHz Bruker Avancespectrometer equipped with 5 mm cryoprobe. The numbering scheme used toidentify the residues is indicated in FIG. 1. GlcNAc_(ext) stands forN-acetylglucosamine in lactosamine extension; Gal_(ext) indicatesgalactose in lactosamine extension.

FIG. 15 shows a 2D ¹H-¹H TOCSY spectrum of a 2AB-labeled N-glycan pool,acquired on a 600 MHz Bruker Avance spectrometer equipped with 5 mmcryoprobe at 25° C. in D₂O. Fucose cross-peaks are indicated.

FIG. 16 is a table showing the chemical shifts for various peaks in thespectra of FIGS. 12 and 14.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

N-linked glycans are glycans that are linked to a glycoconjugate via anitrogen linkage. The diverse assortment of N-glycans are based on thecommon core pentasaccharide (Man)₃(GlcNAc)(GlcNAc) (see FIG. 1). Anexemplary tetraantennary N-glycan is shown in FIG. 2. Typical N-glycansmay vary in the fucosylation of GlcNAc1, the number of branches of theMan4 and 4′ residues, and the sialylation of the branches. Additionally,the sugar residues may be modified, such as by sulfation orphosphorylation. Extensions of the branches are possible by theinsertion of a lactosamine.

For illustrative purposes, FIG. 3 shows the structures of threedifferent model N-glycans, namely A1F which is a monosialo (α2-6)biantennary fucosylated N-glycan, NA3 which is an asialo triantennaryN-glycan in which the Man4′ is monosubstituted, and NA4 which is anasialo tetraantennary N-glycan. The structures of these and otherexemplary N-glycans are compared in FIGS. 4A to 4U. In general,N-glycans may be grouped as “complex” A-4 (tetraantennary, such as NA4and NGA4); A-3 (triantennary, such as A3,NA3, NGA3); A2F (fucosylatedand biantennary, such as A2F, A1F, NA2F, NGA2F); A-2 (biantenarry, suchas A2); “hybrid” and “high mannose” (e.g., Man-5, Man-6, Man-7, Man-8,Man-9) type.

N-linked glycans are commonly found as components of proteins (i.e., aglycoprotein). N-linked glycans are linked to the glycoprotein in theendoplasmic reticulum and the Golgi apparatus via a N-linkage.Typically, glycans are added to the glycoprotein in the lumen of theendoplasmic reticulum. The glycan is added to the amino group on theside chain of an asparagine residue contained within the targetconsensus sequence of Asn-X-Ser/Thr, where X may be any amino acidexcept proline, to provide an N-linked glycan. The initial glycan chainis usually trimmed by specific glycosidase enzymes in the endoplasmicreticulum, resulting in a short, branched core comprised of twoN-acetylglucosamine and three mannose residues. After initial processingin the endoplasmic reticulum, the glycoprotein is then transported tothe Golgi where further processing may take place. The trimmed N-linkedglycan moiety may be modified by the addition of several mannoseresidues, resulting in a ‘high-mannose oligosaccharide’. Additionally oralternatively, one or more monosaccharides units of N-acetylglucosaminemay be added to the core mannose subunits to form ‘complex glycans’.Galactose may be added to the N-acetylglucosamine subunits, and sialicacid subunits may be added to the galactose subunits, resulting in achain that terminates with any of a sialic acid, a galactose or anN-acetylglucosamine residue. Additionally, a fucose residue may be addedto an N-acetylglucosamine residue of the glycan core. Each of theseadditions is catalyzed by specific glycosyl transferases.

As described below, we have used the model N-glycans of FIG. 3 andmixtures of N-glycans to identify features in different types of NMRspectra that can be used to characterize various aspects of N-glycans.In particular, we have identified NMR signals with chemical shifts thatare diagnostic of glycan structural features and that can be resolved inspectra of certain glycan mixtures.

The following sections describe particular 1D ¹H, 2D ¹H-¹H and 2D ¹H-¹³Cexperiments that were used to identify diagnostic NMR signals. It is tobe understood that the methods are in no way limited to the specificpulse sequences or experiments described herein. Thus, any NMR pulsesequence or experiment that is capable of identifying a ¹H chemicalshift, ¹H-¹H scalar correlation, ¹H-¹³C scalar correlation or other NMRsignal that is described herein may be used in a method. In general, itwill be appreciated that the choice of experiment may depend on factorssuch as the specific chemical shift(s) of interest, spectral crowding,amount and nature of sample, desired timeframe, need for quantitativeinformation, etc. It will also be appreciated that the methods are in noway limited to the specific chemical shifts described herein. Inparticular, it is well known that chemical shifts may vary depending onexperimental conditions, e.g., solvent, temperature, etc. Thus, wheneverwe refer to a particular chemical shift herein it is to be understoodthat this is in reference to a particular set of experimentalconditions. Those skilled in the art will be able to determine suitablechemical shifts for NMR signals described herein under differentexperimental conditions.

¹H Chemical Shifts

In one aspect, the present disclosure provides methods which utilize ¹Hchemical shifts to identify a structural characteristic of N-glycans.While these ¹H chemical shifts may be obtained from a simple 1D ¹H NMRspectrum, they may also be obtained from a 2D ¹H-¹H spectrum, a 2D¹H-¹³C spectrum, etc. According to this aspect of the disclosure, asample is provided which includes a mixture of N-glycans. ¹H chemicalshifts in the sample are then obtained according to any method known inthe art. In the Examples we describe the use of 1D ¹H NMR spectra thatwere obtained on a 600 MHz Bruker Avance spectrometer with 5 mmcryoprobe at 27° C. with presaturation of the water resonance. Eachspectrum was obtained by averaging 16 to 256 scans. The recycle delaywas 14 s. It will be appreciated however that for purposes of thisdisclosure, the 1D ¹H spectra may be obtained using higher or lowerfield spectrometers, using different probes, conditions, watersuppression sequence, recycle delay, detector cycling, etc.

We have found that despite the significant spectral crowding in 1D ¹Hspectra, these spectra can provide quantitative information even oncomplex mixtures of N-glycans in D₂O. For purposes of illustration, 1Dspectra of the three model N-glycans of FIG. 3 and of a mixture ofN-glycans are shown in FIG. 5 a-5 c and 5d, respectively. The ¹Hchemical shifts in these spectra provide much structural information.Thus once the ¹H chemical shifts have been obtained (e.g., from a 1D ¹Hspectrum), the methods include a step of identifying whether the ¹Hchemical shifts includes one or more shifts that are associated with astructural characteristic. The outcome of this identification step isthen used to determine whether the sample includes a glycan with thestructural characteristic.

In some embodiments, the specific chemical shift is within the anomericregion (ca. 4.5 ppm to ca. 5.5 ppm) which is the most resolved region ofthe 1D ¹H spectrum (although the residual water signal at ca. 4.8 ppmpartially obscures some of the anomeric signals). For example, we havefound that the presence of oligomannose structures can be detected byanalysis of the anomeric region. Oligomannose residues presentcharacteristic and well resolved anomeric signals at ca. 5.00-5.10 ppmand ca. 5.35-5.45 ppm, as shown with an asterisk (*) in FIG. 6.Additional characteristic signals are described herein.

In some embodiments, the specific signal or signals are in the methylregion (ca. 0.7 ppm to ca. 2.8 ppm). For example, in one embodiment, themethods involve determining whether the spectrum includes a chemicalshift at ca. 2.0 ppm which belongs to the acetyl methyl-¹H signal ofGlcNAc or a sialic acid. In certain embodiments, GlcNAc and sialic acidscan be distinguished on the basis of the axial and equatorial H3 signalsof sialic acids that are readily observed in the range of ca. 1.6 ppm toca. 1.9 ppm and ca. 2.6 ppm to ca. 2.8 ppm, respectively (see FIGS. 5 aand d). In some embodiments, the sialic acid H3 axial signal can be usedas a diagnostic of the linkage type, with α2-3 and α2-6 linkagesresonating at ca. 1.7 ppm and ca. 1.8 ppm, respectively. As discussedbelow, a 2D ¹H-¹H TOCSY spectrum can provide additional resolution bythe well-resolved H3 axial and H3 equatorial cross-peaks.

In certain embodiments, the presence of di- or tri-acetylated NeuAc(e.g., Neu5,9Ac₂) can be identified from a characteristic signal at ca.2.15 ppm, as indicated in FIG. 7 which was obtained with a mixture ofN-glycans.

In some embodiments, the presence of fucose within the sample can bedetermined based on the presence of a methyl-¹H signal at ca. 1.2 ppm(see FIGS. 5 a and d). As discussed below, the core location of thefucosylation can be confirmed by a ¹H-¹³C HSQC spectrum (FIG. 11), inwhich the GlcNAc2 anomeric chemical shift is perturbed by the presenceof a fucose on GlcNAc1. As discussed in more detail below and as shownin FIG. 15, we have shown that fucose ¹H methyl signals can be resolvedwhen fucose occurs in different environments (e.g., in 2AB-labeled andunlabeled glycans). In some embodiments, the sensitivity of the fucose¹H methyl signals to their environment can be used to detect andquantify fucose moieties that are in an antennary environment (e.g., asopposed to a core environment). In some embodiments, this is achievedusing a 2D ¹H-¹H TOCSY or other homonuclear scalar correlationexperiment.

In certain embodiments it may prove advantageous to quantify one or morecharacteristic ¹H signals. Each characteristic signal can be quantifiedby integration. As long as the recycle delay between scans issufficiently long (typically about five times the longitudinalrelaxation time, T₁, of the slowest relaxing species), the integrals arequantitative. Signals within ca. 0.2 ppm to ca. 0.3 ppm of the residualwater signal (ca. 4.8 ppm) will typically be partially attenuated by thesame presaturation used to suppress water and will therefore be lessquantitative than those that are further removed. In certainembodiments, peak fitting software may be used to quantify one or morecharacteristic ¹H signals. Peak fitting software is particularly usefulwhen two peaks are partially overlapping. In certain embodiments,quantitative results may be used to yield ratios based on comparisonswith the results obtained with a different sample (e.g., a calibrationstandard, a different glycan preparation, etc.).

¹H-¹H Scalar Correlations

In another aspect, structural characteristics of N-glycans can beidentified using ¹H-¹H scalar correlations (e.g., without limitation, ina 2D ¹H-¹H TOCSY spectrum). According to this aspect ¹H-¹H scalarcorrelations are detected for the sample of interest and at least onecorrelation is identified which is known to be associated with aparticular structural characteristic. In the Examples we describe, interalia, 2D ¹H-¹H TOCSY spectra that were acquired on a 600 MHz BrukerAvance spectrometer with 5 mm cryoprobe at 27° C. using 120 ms MLEV-17mixing. A total of 4 points were averaged for each of 4096×256hypercomplex points. The recycle delay was 1.4 s. However, it will beappreciated that the 2D ¹H-¹H TOCSY spectrum may be obtained using anyknown pulse sequence and any suitable set of experimental conditions. Ina 2D ¹H-¹H TOCSY experiment, a ‘mixing time’ present within the pulsesequence enables magnetization to be transferred using the scalarcoupling between protons that are closely linked by chemical bonds. Thismagnetization transfer results in ¹H-¹H correlations which are nearlyalways restricted to protons within the same sugar residue. Varying themixing time used to affect the transfer alters the number of bonds overwhich the correlations occur. A 2D ¹H-¹H TOCSY spectrum of a modelN-glycan (A1F, see FIG. 3) is shown in FIG. 8. In certain embodiments,known ¹H-¹H scalar couplings are used to model the magnetizationtransfer and thereby adjust any quantitative information obtained frompeak integrals. Signals close to the water signal will be partiallyattenuated by the presaturation used to suppress water.

As described previously, it is to be understood that any NMR experimentmay be used to identify ¹H-¹H scalar correlations. Thus, withoutlimitation, instead of a 2D ¹H-¹H TOCSY experiment one could use a 1D ¹Hselective TOCSY experiment, COSY, multiple-quantum-filtered variants ofCOSY, isotope-filtered versions of COSY and TOCSY, TOCSY-HSQC,TOCSY-HMQC experiments, etc. Useful experiments also include ROESY andNOESY and their variants, insofar as these dipolar-correlationexperiments can be utilized to elucidate ¹H-¹H correlations within amonosaccharide ring, and can thereby be utilized to elucidate diagnosticpatterns of chemical shifts, pertaining to specific monosaccharide ringstructures. Possible experiments also include any selective onedimensional analog of the two dimensional experiments listed above.

As an illustrative example, as mentioned above, while the presence ofsialylation can be readily identified from the ¹H chemical shifts of theaxial and equatorial H3 signals, ¹H-¹H scalar correlations provideadditional resolution by the location of the well-resolved H3 axial andH3 equatorial cross-peaks, e.g., in a 2D ¹H-¹H TOCSY spectrum (ca. 1.6ppm to ca. 1.9 ppm/ca. 2.6 ppm to ca. 2.8 ppm).

Similarly, ¹H-¹H scalar correlations allows for discrimination betweenthe branching options at the Man4 position as shown in the 2D ¹H-¹HTOCSY spectrum of FIG. 9. The branching of the Man4 residue can bedistinguished on the basis of the location of the H2-H3 cross-peak (ca.4.25 ppm/ca. 3.90 ppm for mono-antennary vs. ca. 4.25 ppm / ca. 4.10 ppmfor bi-antennary).

For example, the chemical shifts of the Man4 cross-peaks may range asfollows:

-   -   mono-antennary: H2: ca. 4.2 to 4.3 ppm and H3: ca. 3.85 to ca.        3.95 ppm    -   bi-antennary: H2: ca. 4.2 to 4.3 ppm and H3: ca. 4.05 to ca.        4.15 ppm

Without limitation, branching at the Man4 position may also bedetermined by using a 1D ¹H selective TOCSY pulse sequence. For example,in various embodiments one can select the Man4 H2 signal at ca. 4.25 ppmand determine whether this leads to transfer of polarization to an H3peak at ca. 3.90 ppm (mono-antennary) or ca. 4.10 ppm (bi-antennary).

1D ¹H selective TOCSY pulse sequences may also be used in other contextsto more clearly assign specific 1D ¹H peaks. For example, the H1 signalof a galactose residue in a lactosamine extension resonates at ca. 4.57ppm in our experiments. When selected using a 1D ¹H selective TOCSYpulse sequence, TOCSY correlations can be used to identify H-¹ H scalarcorrelations within the galactose residue. These ¹H-¹H scalarcorrelations can then be used to confirm the location of the galactoseresidue to be within a polylactosamine extension. It will be appreciatedthat these correlations may alternatively be identified in the contextof a different NMR experiment, e.g., without limitation a 2D ¹H-¹H TOCSYexperiment.

¹H-¹H scalar correlations may also be used to identify the presence of asulfated GlcNAc moiety. Indeed, 6-O-sulfation should give rise to adiagnostic ¹H chemical shift for H6 and other ¹H signals around themonosaccharide ring system. While these ¹H signals may be present withina crowded region of the spectrum, a 2D ¹H- ¹H TOCSY or 1D ¹H selectiveTOCSY experiment can be used to reveal a pattern of ¹H-¹H scalarcorrelations, which, taken together, are diagnostic for the 6-O-sulfatedGlcNAc.

This approach can also be used to identify the presence of aphosphorylated mannose moiety. Indeed, 6-O-phosphorylation should giverise to a diagnostic ¹H chemical shift for H6 and other ¹H signalsaround the monosaccharide ring system. While these ¹H signals may bepresent within a crowded region of the spectrum, the position of thephosphomannose H6 signal can be determined using a ¹H-³¹P scalarcorrelation experiment. The remainder of the phosphomannose spin systemcan then be resolved from the rest of the overlapped portion of thespectrum using a ³¹P -¹H HSQC-TOCSY pulse sequence which selectsmagnetization from ³¹P-¹H and then transfers it to other protons aroundthe phosphomannose ring via a TOCSY sequence. Alternatively, a simple 2D¹H-¹H TOCSY or selective 1D ¹H TOCSY experiment can be used to reveal apattern of ¹H-¹H scalar correlations, which, taken together, arediagnostic for the 6-O-phosphorylated mannose.

It is to be understood that the improvements in resolution that can beobtained using ¹H-¹H scalar correlations will generally apply to any 1D¹H signal where the detected proton possesses a sufficiently strong¹H-¹H scalar coupling with a neighboring proton. Preferably the twocoupled protons have different chemical shifts. It is also to beunderstood that 1D ¹H, 1D ¹H selective TOCSY and 2D ¹H-¹H TOCSY spectracan be used separately or in conjunction according to the methodsdescribed herein.

¹H-¹³C Scalar Correlations

In another aspect, structural characteristics of N-glycans can beidentified using ¹H-¹³C scalar correlations (e.g., without limitation,in a 2D ¹H-¹³C HSQC spectrum). According to this aspect ¹H-¹³C scalarcorrelations are detected for the sample of interest and at least onecorrelation is identified which is known to be associated with aparticular structural characteristic. ¹H-¹³C scalar correlations (e.g.,in a 2D ¹H-¹³C HSQC spectrum) generally provide even more spectralresolution than ¹H-¹H scalar correlations (e.g., in a 2D ¹H-¹H TOCSYspectrum) since different correlations are now also separated in the ¹³Cdimension. In the Examples we describe 2D ¹H-¹³C HSQC spectra that wereacquired on a 600 MHz Bruker Avance spectrometer with 5 mm cryoprobe at27° C. using a sensitivity-enhanced gradient HSQC pulse sequence. Atotal of 16 points were averaged for each of 1024×256 hyper complexpoints. The recycle delay was 1.1 s. However, it will be appreciatedthat the 2D ¹H-¹³C HSQC spectrum may be obtained using any known pulsesequence and any suitable set of experimental conditions. In a 2D ¹H-¹³CHSQC experiment, a ‘magnetization-transfer delay’ present within thepulse sequence enables magnetization to be transferred using the scalarcoupling between ¹H and ¹³C that are closely linked by chemical bonds.However, the sensitivity of the HSQC measurement is lower than 1D ¹H and2D ¹H-¹H TOCSY experiments due to the low natural abundance of ¹³C(about 1%). As a result the data acquisition times for a 2D ¹H-¹³C HSQCexperiment will generally be longer than for a 2D ¹H-¹H TOCSY which willin turn be longer than for a 1D ¹H experiment. It will be appreciatedthat shorter data acquisition times may be used in the event the sampleincludes isotopically enriched N-glycans (i.e., ¹³C enriched N-glycans).

As described previously it is to be understood that any NMR experimentmay be used to identify ¹H-¹³C scalar correlations. Thus, withoutlimitation, instead of a 2D ¹H-¹³C HSQC experiment one could use 2Dselective TOCSY HSQC, HMQC, TOCSY HMQC, accordion-HSQC, accordion-HMQCexperiments, etc.

For example, in various embodiments it may be advantageous to perform a2D selective TOCSY ¹H-¹³C HSQC experiment to resolve individual sugarspin systems when a spectrum is particularly crowded. In one embodimentthis experiment may be used to determine acetylation positions of sialicacids, e.g., by comparing ¹H and ¹³C chemical shifts for H7, H8 and/orH9 with those of free sialic acid. Indeed, any of the 3 hydroxyl groupsin the C7-C9 side-chain (i.e., CH(OH)—CH(OH)—CH(OH)—(CH₂OH) may beacetylated. If acetylation has occurred, this will result in asignificant downfield chemical shift of the CH proton at the acetylationposition. The TOCSY experiment will reveal any such significant changesin the chemical shift of these protons, and will therefore reveal which,if any, of these positions are acetylated.

For illustrative purposes, the 2D ¹H-¹³C HSQC spectrum of a modelN-glycan (A1F) is shown in FIG. 10. 2D ¹H-¹³C HSQC spectra may be usedquantitatively. A correction factor may be required if the ¹H-¹³C scalarcouplings vary significantly between residues. However, signals fromgroups which have similar scalar couplings can be directly comparedwithout correction.

In certain embodiments, the cross-peaks in a 2D ¹H-¹³C HSQC spectrum maybe used to determine the monosaccharide composition of a glycan mixture.In particular, as shown in FIGS. 11, 12 and 14, the anomeric signals foreach residue type produce ¹H-¹³C cross-peaks that are even more resolvedthan in the 1D ¹H spectrum. The anomeric signals of Man4 and Man4′ (withcross-peaks at ¹H/¹³C of ca. 5.15 ppm/ca. 102 ppm and ca. 4.95 ppm/ca.100 ppm, respectively, e.g., see FIG. 11) can be quantified in thismanner. The anomeric signals of GlcNAc and Gal may also be used todetermine the monosaccharide composition of a glycan mixture (e.g., seecross-peaks at ¹H/¹³C of ca. 4.50-4.75 ppm/ca. 104-106 ppm in FIG. 11).It will be appreciated that when cross-peaks from GlcNAc and Galpartially overlap, analytical methods (e.g., peak fitting algorithms)may be used in order to extract quantitative information. Similaranalytical tools may be used in order to compensate for partial signalattenuation caused by presaturation of the neighboring water signal.Sialic acid lacks an anomeric proton, but can be quantified by the axialand equatorial H3 signals in the upfield region of the spectrum (withcross-peaks at ¹H/¹³C of ca. 1.7 ppm/ca. 39 ppm and ca. 2.6 ppm/ca. 39ppm, respectively, data not shown).

Branching at the Man4 and 4′ positions is also readily determined by theanomeric chemical shifts of ¹H-¹³C scalar correlations. For example, asshown by the overlay of 2D ¹H-¹³C HSQC spectra from various modelcompounds in FIG. 11 and the spectrum of an N-glycan pool in FIG. 12,the anomeric signal positions of the branching mannose residues arediagnostic of the number of branches at each position. The equivalentanalysis by simple 1D ¹H analysis is difficult as the Man4 ¹H signalshift between the mono- and di- substituted species is negligible, andthe Man4′ ¹H signal suffers from partial overlap with that of fucose.Nonetheless, as discussed above, characteristic ¹H-¹H scalarcorrelations that are associated with branching at the Man4 position maystill be identified within a 1D spectrum by using a selective pulsesequence, e.g., a 1D ¹H selective TOCSY sequence.

As shown in FIGS. 11 and 12 and summarized in FIG. 16, the anomericchemical shifts of ¹H-¹³C scalar correlations can be used to detect thepresence (or the relative ratio based on relative signal integrals) ofthe following mannose residues and branching patterns in unlabeledN-glycans:

-   -   Man4 mono-antennary: δ_(H) ca. 5.10-5.15 ppm; δ_(c) ca.        102.2-102.6 ppm    -   Man4 bi-antennary: δ_(H) ca. 5.10-5.15 ppm; δ_(c) ca.        101.7-102.1 ppm    -   Man4′ mono-antennary: δ_(H) ca. 4.90-4.95 ppm; δ_(c) ca.        98.8-99.8 ppm    -   Man4′ bi-antennary: δ_(H) ca. 4.83-4.88 ppm; δ_(c) ca.        99.5-100.5 ppm

As shown in FIGS. 11 and 12 and summarized in FIG. 16, the anomericchemical shifts of ¹H-¹³C scalar correlations can be also be used todetect the presence (or the relative ratio based on relative signals) ofthe following core residues in unlabeled N-glycans:

-   -   GlcNAc1α: δ_(H) ca. 5.15-5.20 ppm; δ_(c) ca. 93-94 ppm    -   GlcNAc1β: δ_(H) ca. 4.65-4.70 ppm; δ_(c) ca. 97-98 ppm    -   GlcNac2: δ_(H) ca. 4.63-4.68 ppm; δ_(c) ca. 103.5-104.5 ppm    -   Man3: δ_(H) ca. 4.72-4.77 ppm; δ_(c) ca. 102.7-103.7 ppm

The anomeric chemical shifts of ¹H-¹³C scalar correlations can be alsobe used to detect and/or quantify GlcNacs in β(1-2) linkages to mannose,GlcNacs in β(1-4) or β(1-6) linkages to mannose and GlcNacs inlactosamine-extensions:

-   -   β(1-2) linkages: δ_(H) ca. 4.55-4.60 ppm; δ_(c) ca. 102-103 ppm    -   β(1-4) or β(1-6) linkages: δ_(H) ca. 4.52-4.58 ppm; δ_(c) ca.        104-105 ppm    -   lactosamine-extensions: δ_(H) ca. 4.67-4.72 ppm; δ_(c) ca.        105-106 ppm

The distinction between these different GlcNAc residues can be seen inFIG. 12 by comparing the chemical shifts of GlcNAc5a,c (i.e., β(1-2)linkages to mannose), GlcNAc5b,d (i.e., β(1-4) linkages to mannose) andGlcNac_(ext).

The anomeric chemical shifts of ¹H-¹³C scalar correlations can be alsobe used to detect and/or quantify unsubstitued galactose residues (i.e.,no sialic acid), galactose residues with an α(2-3) sialic acid attached,galactose residues with an α(2-6) sialic acid attached, and galactoseresidues in lactosamine-extensions:

-   -   unsubstitued (no sialic acid): δ_(H) ca. 4.43-4.48 ppm; δ_(c)        ca. 105-106 ppm    -   α(2-3) sialic acid attached: δ_(H) ca. 4.52-4.57 ppm; δ_(c) ca.        105-106 ppm    -   α(2-6) sialic acid attached: δ_(H) ca. 4.41-4.47 ppm; δ_(c) ca.        106-107 ppm    -   lactosamine-extensions: δ_(H) ca. 4.52-4.57 ppm; δ_(c) ca.        105-106 ppm

Peaks for unsubstituted galactose, galactose in a galactosamineextension or galactose with α(2-3) sialic attached (Neu5OAc) are shownin FIG. 12 (the latter pair overlap). A peak for galactose with α(2-6)sialic attached was observed in a 2D ¹H-¹³C HSQC spectrum of the modelglycan A1F-2AB (data not shown).

The anomeric chemical shifts of ¹H-¹³C scalar correlations can be alsobe used to detect and/or quantify oligomannose structures (e.g., in highmannose glycans). Thus, as shown in FIG. 12, in various embodiments,oligomannose structures are associated with one or more ¹H-¹³C scalarcorrelations in the following ranges:

-   -   δ_(H) ca. 5.35-5.45 ppm; δ_(c) ca. 103-104 ppm    -   δ_(H) ca. 5.05-5.15 ppm; δ_(c) ca. 104.5-105.5 ppm    -   δ_(H) ca. 4.95-5.05 ppm; δ_(c) ca. 105-106 ppm

In one embodiment, more than one ¹H-¹³C scalar correlations, e.g., 2 or3 correlations are observed across these ranges. In one embodiment, 1 or2 correlations are observed in the following range:

-   -   δ_(H) ca. 4.90-5.20 ppm; δ_(c) ca. 104-106 ppm

The anomeric chemical shifts of ¹H-¹³C scalar correlations can be alsobe used to detect and/or quantify fucose residues. For example, as shownin FIG. 12, in various embodiments, core fucose residues in unlabeledN-glycans exhibit a correlation in the following anomeric region:

-   -   δ_(H) ca. 4.85-4.95 ppm; δ_(c) ca. 101-103 ppm

Methyl chemical shifts of ¹H-¹³C scalar correlations can be also be usedto detect and/or quantify fucose residues and in particular todistinguish core and antennary fucose residues. For example, in variousembodiments, core and antennary fucose residues in unlabeled N-glycansexhibit a correlation in the following range (data not shown):

-   -   core fucose: δ_(H) ca. 1.17-1.19 ppm; δ_(c) ca. 17.7-18.7 ppm    -   antennary fucose: δ_(H) ca. 1.21-1.24 ppm; δ_(c) ca. 17-19 ppm

In general it is to be understood that the improvement in resolutionthat can be obtained via ¹H-¹³C scalar correlations (e.g., in a 2D¹H-¹³C HSQC spectrum) will generally apply to any 1D ¹H signal where thedetected proton possesses a sufficiently large ¹H-¹³C scalar couplingwith a carbon. It is also to be understood that 1D ¹H chemical shifts,¹H-¹H scalar correlations and ¹H-¹³C scalar correlations can be usedseparately or in conjunction according to the methods described herein.

Analysis of Labeled Glycans

To facilitate the isolation procedure, N-glycan pools are sometimeslabeled, e.g., with a fluorophore. FIG. 14 shows the 2D ¹H-¹³C HSQCspectrum of a 2AB-labeled version of the unlabeled sample that was usedin obtaining the spectrum of FIG. 12. The chemical shifts of the ¹H-¹³Cscalar correlations in FIG. 14 are summarized in FIG. 16. As shown inFIG. 16, chemical shifts for certain residues are shifted as a result ofthe 2AB label, in particular those that are closest to the core (i.e.,closest to the point of attachment of the 2AB label). It will beappreciated that these shifted ranges should be used instead of thosepresented above when analyzing N-glycan mixtures from a 2AB-labeledsample. It will also be appreciated that yet other labels may lead todifferent chemical shift ranges for each residue type and that these arereadily identifiable based on the teachings herein. The presentdisclosure encompasses methods of analyzing these alternatively labeledN-glycans by NMR.

Typically the labeling reaction will cause the N-glycan NMR spectrum tolose a characteristic signal that can be used as a proxy for measuringthe quality and extent of the labeling reaction. For example, FIG. 13shows that 1D ¹H-NMR spectra of N-glycans labeled with 2AB lack signalsdue to GlcNAc1αH1. The level of residual signals due to GlcNAc1αH1 canbe used to demonstrate the effectiveness of the 2AB-labeling procedure.

FIG. 14 shows that the same labeling reaction causes the signals due toGlcNAc1α/β in the 2D ¹H-¹³C HSQC spectrum to disappear. In addition,after 2AB-labeling, Man3 and GlcNAc2 are slightly shifted from theiroriginal position (compare FIG. 12 with FIG. 14). Interestingly, theanomeric fucose signal splits into two partially resolved signals(compare FIG. 12 with FIG. 14). A split is also observed in the 2D¹H-¹³C HSQC spectrum for the methyl fucose signal (data not shown).Thus, in one embodiment, chemical shifts of ¹H-¹³C scalar correlationscan be also be used to detect and/or quantify 2AB-labeled fucoseresidues using correlations in the following ranges:

-   -   core fucose (anomeric): δ_(H′)ca. 4.82-4.88 ppm and δ_(C′)ca.        101-102 ppm δ_(H″)ca. 4.88-4.93 ppm and δ_(c″)ca. 101-102 ppm    -   core fucose (methyl): δ_(H′)ca. 1.15-1.20 ppm and δ_(C′)ca.        17.7-18.7 ppm δ_(H″)ca. 1.20-1.25 ppm and δ_(C″)ca. 17.7-18.7        ppm

The split fucose signals are also clearly resolved in a 2D ¹H-¹H TOCSYexperiment, where two different signals due to fucose H1/H2 (and H1/H3)cross-peaks can be identified (see FIG. 15). Moreover, two separatesignals for fucose —CH₃/H5 cross-peaks are also clearly visible (seeFIG. 15).

Combination With Enzyme Digestion

In one aspect, one or more of the NMR methods described above can beused in combination with enzymatic treatment, e.g., to elucidate thebranching position of a complex glycan.

In some embodiments, the combination of NMR with enzymatic treatments ofglycans enables the location of specific antennae to be determined onthe glycan of interest. For example, if a glycan contains threesialylated antennae and one non-sialylated antenna, enzymatic treatmentscan be used that will remove the non-sialylated antenna. This willresult in a change of the Man4 or Man4′ NMR signals from a biantennaryto a monoantennary pattern. The position of attachment of thenon-sialylated antenna can therefore be determined from the NMR data.

When treating a sample with different enzymes, treatment may besimultaneous or sequential. In one embodiment, NMR data may be obtainedon the sample prior to enzymatic treatment and after each phase oftreatment (e.g., in a sequential experiment). In one embodiment, NMRdata may be obtained continuously during in situ enzymatic treatment. Insitu NMR reduces sample loss and also allows the enzymatic reaction tobe monitored in real time, thereby confirming optimal conditions andduration for enzymatic treatment.

Applications

It will be appreciated that the techniques described herein can beutilized in any of a variety of applications. In general, thesetechniques are useful in any application that involves the structuralcharacterization of N-glycans. Techniques of the present disclosure maybe particularly useful in characterizing monosaccharide composition,branching, fucosylation, sulfation, phosphorylation, sialylationlinkages (α2-3 vs. α2-6), presence of impurities and/or efficiency of alabeling procedure (e.g., labeling with a fluorophore such as 2-AB).

Methods of the present disclosure can be applied to glycan mixturesobtained from a wide variety of sources including, but not limited to,therapeutic formulations and biological samples. A biological sample mayundergo one or more analysis and/or purification steps prior to or afterbeing analyzed according to the present disclosure. To give but a fewexamples, in some embodiments, a biological sample is treated with oneor more proteases and/or glycosidases (e.g., so that glycans arereleased); in some embodiments, glycans in a biological sample arelabeled with one or more detectable markers or other agents that mayfacilitate analysis by, for example, mass spectrometry or NMR. Any of avariety of separation and/or isolation steps may be applied to abiological sample in accordance with the present disclosure.

Methods of the present disclosure can be utilized to analyze glycans inany of a variety of states including, for instance, free glycans,glycoconjugates (e.g., glycopeptides, glycolipids, proteoglycans, etc.),or cells or cell components, etc. In one embodiment, the methods areused to analyze a glycan preparation. In one embodiment, the methods areused to analyze a glycoprotein preparation.

Methods of the present disclosure may be used in one or more stages ofprocess development for the production of a therapeutic or othercommercially relevant glycoprotein of interest. Non-limiting examples ofsuch process development stages that can employ methods of the presentdisclosure include cell selection, clonal selection, media optimization,culture conditions, process conditions, and/or purification procedure.Those of ordinary skill in the art will be aware of other processdevelopment stages.

The methods can also be utilized to monitor the extent and/or type ofglycosylation occurring in a particular cell culture, thereby allowingadjustment or possibly termination of the culture in order, for example,to achieve a particular desired glycosylation pattern or to avoiddevelopment of a particular undesired glycosylation pattern.

The methods can also be utilized to assess glycosylation characteristicsof cells or cell lines that are being considered for production of aparticular desired glycoprotein (for example, even before the cells orcell lines have been engineered to produce the glycoprotein, or toproduce the glycoprotein at a commercially relevant level).

In some embodiments of the disclosure, a desired glycosylation patternfor a particular target glycoprotein (e.g., a cell surface glycoprotein)is known, and the technology described herein allows monitoring ofculture samples to assess progress of the production along a route knownto produce the desired glycosylation pattern. For example, where thetarget glycoprotein is a therapeutic glycoprotein, for example havingundergone regulatory review in one or more countries, it will often bedesirable to monitor cultures to assess the likelihood that they willgenerate a product with a glycosylation pattern as close to theestablished glycosylation pattern of the pharmaceutical product aspossible, whether or not it is being produced by exactly the same route.As used herein, “close” refers to a glycosylation pattern having atleast about a 75%, 80%, 85%, 90%, 95%, 98%, or 99% correlation to theestablished glycosylation pattern of the pharmaceutical product. In suchembodiments, samples of the production culture are typically taken atmultiple time points and are compared with an established standard orwith a control culture in order to assess relative glycosylation.

In some embodiments, a desired glycosylation pattern will be moreextensive. For example, in some embodiments, a desired glycosylationpattern shows high (e.g., greater than about 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more) occupancy of glycosylation sites; in someembodiments, a desired glycosylation pattern shows, a high degree ofbranching (e.g., greater than about 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or more have tri or tetraantennary structures).

In some embodiments, a desired glycosylation pattern will be lessextensive. For example, in some embodiments, a desired glycosylationpattern shows low (e.g., less than about 35%, 30%, 25%, 20%, 15% orless) occupancy of glycosylation sites; and/or a low degree of branching(e.g., less than about 20%, 15%, 10%, 5%, or less have tri ortetraantennary structures).

In some embodiments, a desired glycosylation pattern will be moreextensive in some aspects and less extensive than others. For example,it may be desirable to employ a cell line that tends to produceglycoproteins with long, unbranched oligosaccharide chains.Alternatively, it may be desirable to employ a cell line that tends toproduce glycoproteins with short, highly branched oligosaccharidechains.

In some embodiments, a desired glycosylation pattern will be enrichedfor a particular type of glycan structure. For example, in someembodiments, a desired glycosylation pattern will have low levels (e.g.,less than about 20%, 15%, 10%, 5%, or less) of high mannose or hybridstructures, high (e.g., more than about 60%, 65%, 70%, 75%, 80%, 85%,90% or more) levels of high mannose structures, or high (e.g., more thanabout 60%, 65%, 70%, 75%, 80%, 85%, 90% or more; for example at leastone per glycoprotein) or low (e.g., less than about 20%, 15%, 10%, 5%,or less) levels of phosphorylated high mannose.

In some embodiments, a desired glycosylation pattern will include atleast about one sialic acid. In some embodiments, a desiredglycosylation pattern will include a high (e.g., greater than about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more) level of termini that aresialylated. In some embodiments, a desired glycosylation pattern thatincludes sialylation will show at least about 85%, 90%, 95% or moreN-acetylneuraminic acid and/or less than about 15%, 10%, 5% or lessN-glycolylneuraminic acid.

In some embodiments, a desired glycosylation pattern shows specificityof branch elongation (e.g., greater than about 50%, 55%, 60%, 65%, 70%or more of extension is on α1,6 mannose branches, or greater than about50%, 55%, 60%, 65%, 70% or more of extension is on α1,3 mannosebranches).

In some embodiments, a desired glycosylation pattern will include a low(e.g., less than about 20%, 15%, 10%, 5%, or less) or high (e.g., morethan about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more) level ofcore fucosylation.

Whether or not monitoring production of a particular target protein forquality control purposes, the methods may be utilized, for example, tomonitor glycosylation at particular stages of development, or underparticular growth conditions.

In some particular embodiments, methods described herein can be used tocharacterize and/or control or compare the quality of therapeuticproducts. To give but one example, the present methodologies can be usedto assess glycosylation in cells producing a therapeutic proteinproduct. Particularly given that glycosylation can often affect theactivity, bioavailability, or other characteristics of a therapeuticprotein product, methods for assessing cellular glycosylation duringproduction of such a therapeutic protein product are particularlydesirable. Among other things, the methods can facilitate real timeanalysis of glycosylation in production systems for therapeuticproteins.

Representative therapeutic glycoprotein products whose production and/orquality can be monitored in accordance with the present disclosureinclude, for example, any of a variety of hematologic agents (including,for instance, erythropoietins, blood-clotting factors, etc.),interferons, colony stimulating factors, antibodies, enzymes, andhormones.

Representative commercially available glycoprotein products include, forexample:

Protein Product Reference Drug interferon gamma-1b Actimmune ®alteplase; tissue plasminogen activator Activase ®/Cathflo ® Recombinantantihemophilic factor Advate human albumin Albutein ® laronidaseAldurazyme ® interferon alfa-N3, human leukocyte derived Alferon N ®human antihemophilic factor Alphanate ® virus-filtered human coagulationfactor IX AlphaNine ® SD Alefacept; recombinant, dimeric fusion proteinAmevive ® LFA3-Ig bivalirudin Angiomax ® darbepoetin alfa Aranesp ™bevacizumab Avastin ™ interferon beta-1a; recombinant Avonex ®coagulation factor IX BeneFix ™ Interferon beta-1b Betaseron ®Tositumomab Bexxar ® antihemophilic factor Bioclate ™ human growthhormone BioTropin ™ botulinum toxin type A Botox ® alemtuzumab Campath ®acritumomab; technetium-99 labeled CEA-Scan ® alglucerase; modified formof beta- Ceredase ® glucocerebrosidase imiglucerase; recombinant form ofbeta- Cerezyme ® glucocerebrosidase crotalidae polyvalent immune Fab,ovine CroFab ™ digoxin immune Fab, ovine DigiFab ™ rasburicase Elitek ®etanercept Enbrel ® epoietin alfa Epogen ® cetuximab Erbitux ™algasidase beta Fabrazyme ® urofollitropin Fertinex ™ follitropin betaFollistim ™ teriparatide Forteo ® human somatropin GenoTropin ® glucagonGlucaGen ® follitropin alfa Gonal-F ® antihemophilic factor Helixate ®Antihemophilic Factor; Factor XIII Hemofil ® insulin Humalog ®antihemophilic factor/von Willebrand factor Humate-P ® complex-humansomatotropin Humatrope ® adalimumab HUMIRA ™ human insulin Humulin ®recombinant human hyaluronidase Hylenex ™ interferon alfacon-1Infergen ® Eptifibatide Integrilin ™ alpha-interferon Intron A ®palifermin Kepivance anakinra Kineret ™ antihemophilic factor Kogenate ®FS insulin glargine Lantus ® granulocyte macrophage colony-stimulatingLeukine ®/Leukine ® Liquid factor lutropin alfa, for injection LuverisOspA lipoprotein LYMErix ™ ranibizumab Lucentis ® gemtuzumab ozogamicinMylotarg ™ galsulfase Naglazyme ™ nesiritide Natrecor ® pegfilgrastimNeulasta ™ oprelvekin Neumega ® filgrastim Neupogen ® fanolesomabNeutroSpec ™ (formerly LeuTech ®) somatropin [rDNA]Norditropin ®/Norditropin Nordiflex ® insulin; zinc suspension; NovolinL ® insulin; isophane suspension NovolinN ® insulin, regular; NovolinR ® insulin Novolin ® coagulation factor VIIa NovoSeven ® somatropinNutropin ® immunoglobulin intravenous Octagam ® PEG-L-asparaginaseOncaspar ® abatacept, fully human soluable fusion protein Orencia ™muromomab-CD3 Orthoclone OKT3 ® human chorionic gonadotropin Ovidrel ®peginterferon alfa-2a Pegasys ® pegylated version of interferon alfa-2bPEG-Intron ™ Abarelix (injectable suspension); gonadotropin- Plenaxis ™releasing hormone antagonist epoietin alfa Procrit ® aldesleukinProleukin, IL-2 ® somatrem Protropin ® dornase alfa Pulmozyme ®Efalizumab; selective, reversible T-cell blocker Raptiva ™ combinationof ribavirin and alpha interferon Rebetron ™ Interferon beta 1a Rebif ®antihemophilic factor Recombinate ® rAHF/ntihemophilic factor ReFacto ®lepirudin Refludan ® infliximab Remicade ® abciximab ReoPro ™ reteplaseRetavase ™ rituximab Rituxan ™ interferon alfa-2a Roferon-A ® somatropinSaizen ® synthetic porcine secretin SecreFlo ™ basiliximab Simulect ®eculizumab Soliris ® pegvisomant Somavert ® Palivizumab; recombinantlyproduced, Synagis ™ humanized mAb thyrotropin alfa Thyrogen ®tenecteplase TNKase ™ natalizumab Tysabri ® human immune globulinintravenous 5% and Venoglobulin-S ® 10% solutions interferon alfa-n1,lymphoblastoid Wellferon ® drotrecogin alfa Xigris ™ Omalizumab;recombinant DNA-derived Xolair ® humanized monoclonal antibody targetingimmunoglobulin-E daclizumab Zenapax ® ibritumomab tiuxetan Zevalin ™Somatotropin Zorbtive ™ (Serostim ®)

In some embodiments, the disclosure provides methods in which glycansfrom different sources or samples are compared with one another. Incertain embodiments, the disclosure provides methods used to monitor theextent and/or type of glycosylation occuring in different cell cultures.In some such examples, multiple samples from the same source areobtained over time, so that changes in glycosylation patterns (andparticularly in cell surface glycosylation patterns) are monitored. Insome embodiments, one of the samples is a historical sample or a recordof a historical sample. In some embodiments, one of the samples is areference sample. For example, in certain embodiments, methods areprovided herein which can be used to monitor the extent and/or type ofglycosylation occurring in different cell cultures.

In some embodiments, glycans from different cell culture samplesprepared under conditions that differ in one or more selected parameters(e.g., cell type, culture type [e.g., continuous feed vs batch feed,etc.], culture conditions [e.g., type of media, presence orconcentration of particular component of particular medium(a),osmolarity, pH, temperature, timing or degree of shift in one or morecomponents such as osmolarity, pH, temperature, etc.], culture time,isolation steps, etc.) but are otherwise identical, are compared, sothat effects of the selected parameter(s) on glycosylation patterns aredetermined. In certain embodiments, glycans from different cell culturesamples prepared under conditions that differ in a single selectedparameter are compared so that effect of the single selected parameteron glycosylation patterns is determined. Among other applications,therefore, use of techniques as described herein may facilitatedetermination of the effects of particular parameters on glycosylationpatterns in cells.

In some embodiments, glycans from different batches of a glycoprotein ofinterest (e.g., a therapeutic glycoprotein), whether prepared by thesame method or by different methods, and whether prepared simultaneouslyor separately, are compared. In such embodiments, the methods facilitatequality control of glycoprotein preparation. Alternatively oradditionally, some such embodiments facilitate monitoring of progress ofa particular culture producing a glycoprotein of interest (e.g., whensamples are removed from the culture at different time points and areanalyzed and compared to one another). In any of these embodiments,features of the glycan analysis can be recorded, for example in aquality control record. As indicated above, in some embodiments, acomparison is with a historical record of a prior or standard batchand/or with a reference sample of glycoprotein.

In certain embodiments, the methods may be utilized in studies to modifythe glycosylation characteristics of a cell, for example to establish acell line and/or culture conditions with one or more desirableglycosylation characteristics. Such a cell line and/or cultureconditions can then be utilized, if desired, for production of aparticular target glycoconjugate (e.g., glycoprotein) for which suchglycosylation characteristic(s) is/are expected to be beneficial.

In certain embodiments, techniques of the present disclosure are appliedto glycans that are present on the surface of cells. In some suchembodiments, the analyzed glycans are substantially free ofnon-cell-surface glycans. In some such embodiments, the analyzedglycans, when present on the cell-surface, are present in the context ofone or more cell-surface glycoconjugates (e.g., glycoproteins orglycolipids).

In some particular embodiments, cell-surface glycans are analyzed inorder to assess glycosylation of one or more target glycoproteins ofinterest, particularly where such target glycoproteins are notcell-surface glycoproteins. Such embodiments can allow one to monitorglycosylation of a target glycoprotein without isolating theglycoprotein itself. In certain embodiments, the present disclosureprovides methods of using cell-surface glycans as a readout of or proxyfor glycan structures on an expressed glycoprotein of interest. Incertain embodiments, such methods include, but are not limited to, postprocess, batch, screening or “in line” measurements of product quality.Such methods can provide for an independent measure of the glycosylationpattern of a produced glycoprotein of interest using a byproduct of theproduction reaction (e.g., the cells) without requiring the use ofdestruction of any produced glycoprotein. Furthermore, methods of thepresent disclosure can avoid the effort required for isolation ofproduct and the potential selection of product glycoforms that may occurduring isolation.

In certain embodiments, techniques of the present disclosure are appliedto glycans that are secreted from cells. In some such embodiments, theanalyzed glycans are produced by cells in the context of aglycoconjugate (e.g., a glycoprotein or glycolipid).

According to the present disclosure, techniques described herein can beused to detect desirable or undesirable glycans, for example to detector quantify the presence of one or more contaminants in a product, or todetect or quantify the presence of one or more active or desiredspecies.

In various embodiments the methods can be used to detect biomarkersindicative of, e.g., a disease state, prior to the appearance ofsymptoms and/or progression of the disease state to an untreatable orless treatable condition, by detecting one or more specific glycanswhose presence or level (whether absolute or relative) may be correlatedwith a particular disease state (including susceptibility to aparticular disease) and/or the change in the concentration of suchglycans over time.

In certain embodiments, methods described herein facilitate detection ofglycans that are present at very low levels in a source (e.g., abiological sample), e.g., at levels no more than 10%, 8%, 6%, 4%, 2% or1% of the sample composition). In such embodiments, it is possible todetect and/or optionally quantify the levels of glycans comprisingbetween 0.1% and 5%, e.g., between 0.1% and 2%, e.g., between 0.1% and1% of a glycan preparation. In certain embodiments, it is possible todetect and/or optionally quantify the levels of glycans at between about0.1 fmol to about 1 mmol.

In some embodiments, techniques described herein may be combined withone or more other technologies for the detection, analysis, and orisolation of glycans or glycoconjugates. For example, withoutlimitation, the glycans may be separated by any chromatographictechnique prior to analysis. The glycans may be further analyzed by adifferent technique, e.g., mass spectrometry.

In some embodiments, the glycans can be analyzed by chromatographicmethods, including but not limited to, liquid chromatography (LC), highperformance liquid chromatography (HPLC), ultra performance liquidchromatography (UPLC), thin layer chromatography (TLC), amide columnchromatography, and combinations thereof

In some embodiments, the glycans can be analyzed by mass spectrometry(MS) and related methods, including but not limited to, tandem MS,LC-MS, LC-MS/MS, matrix assisted laser desorption ionisation massspectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ionmobility separation with mass spectrometry (IMS-MS), electron transferdissociation (ETD-MS), and combinations thereof

In some embodiments, the glycans can be analyzed by electrophoreticmethods, including but not limited to, capillary electrophoresis (CE),CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gelelectrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)followed by Western blotting using antibodies that recognize specificglycan structures, and combinations thereof.

The methods will be more specifically illustrated with reference to thefollowing examples. However, it should be understood that the methodsare not limited by these examples in any manner.

EXAMPLES Example 1

1D ¹H spectra of (a) A1F, (b) NA3, (c) NA4 and (d) a mixture ofN-glycans were acquired on a 600 MHz Bruker Avance spectrometer with 5mm cryoprobe at 27° C. with presaturation of the water resonance. Thestructures of A1F, NA3 and NA4 are shown in FIG. 2. Each spectrum wasobtained by signal averaging 16 to 256 scans. The recycle delay was 14s. The resulting 1D ¹H spectra are shown in FIG. 5.

Example 2

The 1D ¹H spectra of mixtures of N-glycans were acquired on a 600 MHzBruker Avance spectrometer with 5 mm cryoprobe, at 25° C., withpresaturation of the water resonance. FIG. 6 shows the anomeric regionof one such spectrum. Potential oligomannose structures are indicatedwith an asterisk (*). FIG. 7 shows the methyl region of another suchspectrum. Specific assignments are indicated.

Example 3

The 2D ¹H-¹H TOCSY spectrum of the model N-glycans A1F, NA3 and NA4 (seeFIG. 3) were acquired on a 600 MHz Bruker Avance spectrometer with 5 mmcryoprobe at 27° C. using 120 ms MLEV-17 mixing. A total of 4 pointswere averaged for each of 4096×256 hypercomplex points. The recycledelay was 1.4 s. The resulting spectrum for A1F is shown in FIG. 8. FIG.9 shows the overlaid 2D ¹H-¹H TOCSY spectra of the anomeric regions ofA1F (black), NA3 (red), and NA4 (green). The H2/H3 cross-peak positionof Man4 is diagnostic of branching, with the only monosubstitutedspecies, A1F, showing a distinct shift from the others.

Example 5

The 2D ¹H-¹³C HSQC spectrum of the model N-glycan A1F, NA3 and NA4 (seeFIG. 3) were acquired on a 600 MHz Bruker Avance spectrometer with 5 mmcryoprobe at 27° C. using a sensitivity-enhanced gradient HSQC pulsesequence. A total of 16 points were averaged for each of 1024×256 hypercomplex points. The recycle delay was 1.1 s. The resulting spectrum forA1F is shown in FIG. 10. FIG. 11 shows the overlaid 2D ¹H-¹³C HSQCspectra of the anomeric regions of A1F (black), NA3 (red), and NA4(green).

Example 6

The 2D ¹H-¹³C HSQC spectrum of an N-glycan pool was recorded at 27° C.,in D₂O, with a 600 MHz Bruker Avance spectrometer with 5 mm cryoprobe.The resulting spectrum is shown in FIG. 12. The numbering scheme used toidentify the residues is indicated in FIG. 2. GlcNAc_(ext) stands forN-acetylglucosamine in lactosamine extension; Gal_(ext) indicatesgalactose in lactosamine extension. An asterisk (*) indicates signalsassigned to oligomannose structures. FIG. 16 provides the chemicalshifts for various peaks in the spectrum of FIG. 12.

Example 7

The 1D ¹H spectra of unlabeled and 2-AB labeled N-glycan pools wererecorded at 600MHz, 25° C., in D₂O. FIG. 13 compares the anomeric regionof these spectra. The spectrum of N-glycans labeled with 2-AB lacksignals due to GlcNAc1αH1.

Example 8

The 2D ¹H-¹³C HSQC spectrum of a 2AB-labeled N-glycan pool was recordedat 27° C., in D₂O, with a 600 MHz Bruker Avance spectrometer equippedwith 5 mm cryoprobe. The resulting spectrum is shown in FIG. 14. Thenumbering scheme used to identify the residues is indicated in FIG. 2.GlcNAc_(ext) stands for N-acetylglucosamine in lactosamine extension;Gal_(ext) indicates galactose in lactosamine extension. FIG. 16 providesthe chemical shifts for various peaks in the spectrum of FIG. 14.

Example 9

The 2D ¹H-¹H TOCSY spectrum of a 2AB-labeled N-glycan pool was acquiredon a 600 MHz Bruker Avance spectrometer equipped with 5 mm cryoprobe at25° C. in D₂O. The resulting spectrum is shown in FIG. 15. Fucosecross-peaks are indicated.

Equivalents

All literature and similar material cited in this application,including, but not limited to, patents, patent applications, articles,books, treatises, and web pages, regardless of the format of suchliterature and similar materials, are expressly incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the methods have been described in conjunction with variousembodiments and examples, it is not intended that the methods be limitedto such embodiments or examples. On the contrary, the present disclosureencompasses various alternatives, modifications, and equivalents, aswill be appreciated by those of skill in the art.

While the methods have been particularly shown and described withreference to specific illustrative embodiments, it should be understoodthat various changes in form and detail may be made without departingfrom the spirit and scope of the present disclosure. Therefore, allembodiments that come within the scope and spirit of the presentdisclosure, and equivalents thereto, are intended to be claimed. Theclaims, descriptions and diagrams of the methods, systems, and assays ofthe present disclosure should not be read as limited to the describedorder of elements unless stated to that effect.

1-91. (canceled)
 92. A method for performing a quality control of atherapeutic glycoprotein preparation by NMR comprising steps of:obtaining a glycan preparation from a sample therapeutic glycoproteinpreparation; obtaining an NMR spectrum for a sample of the glycanpreparation; identifying whether the spectrum includes an NMR signalthat is associated with a structural characteristic of an N-glycanassociated with a desired glycosylation pattern for a target therapeuticglycoprotein, wherein: (i) the structural characteristic is anoligomannose structure and the step of identifying comprises determiningwhether the sample produces a ¹H signal with a chemical shift in therange of ca. 4.5 ppm to ca. 5.5 ppm; (ii) the structural characteristicis a GlcNAc or sialic acid residue and the step of identifying comprisesdetermining whether the sample produces a ¹H signal corresponding to anacetyl methyl nucleus of a GlcNAc or sialic acid residue; (iii) thestructural characteristic is a sialic acid residue and the step ofidentifying comprises determining whether the sample produces a ¹Hsignal corresponding to an axial or equatorial H3 nucleus of a sialicacid residue; (iv) the structural characteristic is a sialic acidresidue with an α2-3 linkage and the step of identifying comprisesdetermining whether the sample produces a ¹H signal corresponding to anaxial H3 nucleus of a sialic acid residue; (v) the structuralcharacteristic is a sialic acid residue with an α2-6 linkage and thestep of identifying comprises determining whether the sample produces a¹H signal corresponding to an axial H3 nucleus of a sialic acid residue;(vi) the structural characteristic is di- or tri-acetylated NeuAc andthe step of identifying comprises determining whether the sampleproduces a ¹H signal with a chemical shift at ca. 2.15 ppm; (vii) thestructural characteristic is a fucose residue and the step ofidentifying comprises determining whether the sample produces a ¹Hsignal corresponding to a methyl nucleus of a fucose residue; (viii) thestructural characteristic is a sialic acid and the step of identifyingcomprises determining whether the sample produces a ¹H-¹H scalarcorrelation between the H3 axial and H3 equatorial nuclei of a sialicacid; (ix) wherein the structural characteristic is a mono-antennaryMan4 residue and the step of identifying comprises determining whetherthe sample produces a ¹H-¹H scalar correlation between the H2 and H3nuclei of a Man4 residue; (x) the structural characteristic is abi-antennary Man4 residue and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹H scalar correlationbetween the H2 and H3 nuclei of a Man4 residue; (xi) the structuralcharacteristic is a galactose residue in a lactosamine extension and thestep of identifying comprises determining the chemical shifts of a ¹H-¹Hscalar correlation between the H1 nucleus and another nucleus of agalactose residue; (xii) the structural characteristic is a sulfatedGlcNac residue and the step of identifying comprises determining thechemical shifts of a ¹H-¹H scalar correlation between the H6 nucleus andanother nucleus of a GlcNac residue; (xii) the structural characteristicis a phosphorylated mannose residue and the step of identifyingcomprises determining the chemical shifts of a ¹H-¹H scalar correlationbetween the H6 nucleus and another nucleus of a mannose residue; (xiv)the structural characteristic is a core fucose residue and the step ofidentifying comprises determining the chemical shifts of a ¹H-¹³C scalarcorrelation for the anomeric nucleus of a GlcNAc2 residue; (xv) thestructural characteristic is a sialic acid residue and the step ofidentifying comprises determining whether the sample produces a ¹H-¹³Cscalar correlation with chemical shifts corresponding to an axial H3nucleus of a sialic acid residue; (xvi) the structural characteristic isa sialic acid residue and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an equatorial H3 nucleus of a sialic acidresidue; (xvii) the structural characteristic is an acetylated sialicacid residue and the step of identifying comprises determining the ¹Hchemical shift of a ¹H-¹³C scalar correlation of the H7, H8 and/or H9nuclei of a sialic acid residue; (xviii) the structural characteristicis a Man4 residue and the step of identifying comprises determining thechemical shifts of a ¹H-¹³C scalar correlation corresponding to ananomeric nucleus of a Man4 residue; (xix) the structural characteristicis a mono- or bi-antennary Man4 residue and the step of determiningcomprises determining whether the Man4 residue is mono-antennary orbi-antennary based on the chemical shifts of the ¹H-¹³C scalarcorrelation; (xx) the structural characteristic is a Man4′ residue andthe step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto an anomeric nucleus of a Man4′ residue; (xxi) the structuralcharacteristic is a mono- or bi-antennary Man4′ residue and the step ofdetermining comprises determining whether the Man4′ residue ismono-antennary or bi-antennary based on the chemical shifts of the¹H-¹³C scalar correlation; (xxii) the structural characteristic is aGlcNAc1 residue and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an anomeric nucleus of a GlcNAc1 residue;(xxiii) the structural characteristic is a GlcNAc2 residue and the stepof identifying comprises determining whether the sample produces a¹H-¹³C scalar correlation with chemical shifts corresponding to ananomeric nucleus of a GlcNAc2 residue; (xxiv) the structuralcharacteristic is a Man3 residue and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to an anomeric nucleus of a Man3 residue;(xxv) the structural characteristic is a GlcNac residue with a β(1-2)linkage to mannose and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an anomeric nucleus of a GlcNac residue with aβ(1-2) linkage to mannose; (xxvi) the structural characteristic is aGlcNAc residue with a β(1-4) or β(1-6) linkage to mannose and the stepof identifying comprises determining whether the sample produces a¹H-¹³C scalar correlation with chemical shifts corresponding to ananomeric nucleus of a GlcNAc residue with a β(1-4) or β(1-6) linkage tomannose; (xxvii) the structural characteristic is a GlcNAc residue in alactosamine extension and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an anomeric nucleus of a GlcNac residue in alactosamine extension; (xxviii) the structural characteristic is anunsubstituted galactose residue and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to an anomeric nucleus of an unsubstitutedgalactose residue; (xxix) the structural characteristic is a galactoseresidue with an α(2-3) sialic acid attached and the step of identifyingcomprises determining whether the sample produces a ¹H-¹³C scalarcorrelation with chemical shifts corresponding to an anomeric nucleus ofa galactose residue with an α(2-3) sialic acid attached; (xxx) thestructural characteristic is a galactose residue with an α(2-6) sialicacid attached and the step of identifying comprises determining whetherthe sample produces a ¹H-¹³C scalar correlation with chemical shiftscorresponding to an anomeric nucleus of a galactose residue with anα(2-6) sialic acid attached; (xxxi) the structural characteristic is agalactose residue in a lactosamine extension and the step of identifyingcomprises determining whether the sample produces a ¹H-¹³C scalarcorrelation with chemical shifts corresponding to an anomeric nucleus ofa galactose residue in a lactosamine extension; (xxxii) the structuralcharacteristic is an oligomannose structure and the step of identifyingcomprises determining whether the sample produces a ¹H-¹³C scalarcorrelation with chemical shifts corresponding to an anomeric nucleus ofan oligomannose structure; (xxxiii) the structural characteristic is acore fucose residue and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an anomeric nucleus of a core fucose residue;(xxxiv) the structural characteristic is a core fucose residue and thestep of identifying comprises determining whether the sample produces a¹H-¹³C scalar correlation with chemical shifts corresponding to a methylnucleus of a core fucose residue; (xxxv) the structural characteristicis an antennary fucose residue and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to a methyl nucleus of an antennary fucoseresidue; (xxxvi) the structural characteristic is a label attached toGlcNAc1 and the step of identifying comprises determining whether thesample produces a ¹H signal corresponding to GlcNAc1αH1; (xxxvii) thestructural characteristic is a label attached to GlcNAc1 and the step ofidentifying comprises determining whether the sample produces a split¹H-¹H scalar correlation corresponding to fucose H1/H2, H1/H3 or —CH₃/H5nuclei; (xxxviii) the structural characteristic is a label attached toGlcNAc1 and the step of identifying comprises determining whether thesample produces a ¹H-¹³C scalar correlation corresponding to an anomericnucleus of GlcNAc1α or GlcNAc1β; (xxxix) the structural characteristicis a label attached to GlcNAc1 and the step of identifying comprisesdetermining the chemical shifts of a ¹H-¹³C scalar correlationcorresponding to an anomeric nucleus of a GlcNAc2 or Man3 residue;(xxxx) the structural characteristic is a label attached to GlcNAc1 andthe step of identifying comprises determining whether the sampleproduces a split ¹H-¹³C scalar correlation corresponding to an anomericfucose nucleus; or (xxxxi) the structural characteristic is a labelattached to GlcNAc1 and the step of identifying comprises determiningwhether the sample produces a split ¹H-¹³C scalar correlationcorresponding to a methyl fucose nucleus; and optionally quantifying theNMR signal if the spectrum includes the signal; and recording the resultof the identification or quantification steps in a quality controlrecord for the sample therapeutic glycoprotein preparation if thespectrum includes the signal.
 93. The method of claim 92, wherein thetarget therapeutic glycoprotein is a therapeutic antibody and the sampletherapeutic glycoprotein preparation is a sample therapeutic antibodypreparation.
 94. The method of claim 92, wherein the target therapeuticglycoprotein is a therapeutic enzyme and the sample therapeuticglycoprotein preparation is a sample therapeutic enzyme preparation. 95.The method of claim 92, wherein the target therapeutic glycoprotein is atherapeutic interferon and the sample therapeutic glycoproteinpreparation is a sample therapeutic interferon preparation.
 96. Themethod of claim 92, wherein the target therapeutic glycoprotein is atherapeutic hematologic agent and the sample therapeutic glycoproteinpreparation is a sample therapeutic hematologic agent preparation. 97.The method of claim 92, wherein the target therapeutic glycoprotein is atherapeutic hormone and the sample therapeutic glycoprotein preparationis a sample therapeutic hormone preparation.
 98. The method of claim 92,wherein the target therapeutic glycoprotein is a therapeutic colonystimulating factor and the sample therapeutic glycoprotein preparationis a sample therapeutic colony stimulating factor preparation.
 99. Themethod of claim 92, further comprising quantifying the NMR signal. 100.The method of claim 92, further comprising quantifying the amount ofN-glycans in the sample that have the structural characteristic. 101.The method of claim 92, further comprising comparing the result of theidentifying or quantifying with a reference sample of the targettherapeutic glycoprotein.
 102. The method of claim 92, furthercomprising steps of quantifying the NMR signal; quantifying the amountof N-glycans in the sample that have the structural characteristic;comparing the result of the identifying or quantifying with a referencesample of the target therapeutic glycoprotein; and recording the resultof the comparing in a quality control record for the sample therapeuticglycoprotein preparation.
 103. The method of claim 92, wherein the stepof obtaining an NMR spectrum comprises using a magnet having a strengthof at least 600 MHz.
 104. The method of claim 92, further comprising astep of obtaining a signal integral within the NMR spectrum.
 105. Themethod of claim 104, wherein the signal integral is obtained bymeasuring signal intensity.
 106. The method of claim 104, wherein thesignal integral is obtained by measuring signal area.
 107. The method ofclaim 92, wherein the sample therapeutic glycoprotein preparationcomprises glycans in a state selected from the group consisting of freeglycans, glycoconjugates, and cells.
 108. The method of claim 92,wherein the step of obtaining a glycan preparation comprises subjectingthe sample therapeutic glycoprotein preparation to enzyme digestion sothat glycans are released from glycoproteins in the glycoproteinpreparation.
 109. The method of claim 92, wherein the step of obtaininga glycan preparation comprises obtaining a cell line that expresses atherapeutic glycoprotein of interest.
 110. The method of claim 109,wherein the therapeutic glycoprotein of interest is not naturallyproduced by the cell.
 111. The method of claim 109, further comprisingsteps of comparing the NMR spectrum of the sample to an NMR spectrum ofa reference sample, wherein the reference sample has an establishedglycosylation characteristic; and based on the comparison, assessing thelikelihood that the cells will generate the therapeutic glycoprotein ofinterest with a glycosylation characteristic close to the establishedglycosylation characteristic of the reference sample.
 112. The method ofclaim 92, wherein the step of identifying comprises obtaining one ormore of a 2D NMR spectrum of the sample; a 2D ¹H-¹H TOCSY NMR spectrumof the sample; a 1D selective ¹H TOCSY NMR spectrum of the sample; or a2D ¹H-¹³C HSQC NMR spectrum of the sample.
 113. The method of claim 92,wherein the structural characteristic is a label attached to an N-glycanand the step of identifying comprises determining whether the sampleproduces a scalar correlation corresponding to said N-glycan.
 114. Themethod of claim 92, further comprising steps of treating the sample witha digestive enzyme to produce a digested sample and repeating the stepof identifying with the digested sample.
 115. The method of claim 92,wherein the structural characteristic is an oligomannose structure andthe step of identifying comprises determining whether the sampleproduces a ¹H signal with a chemical shift in the range of ca. 4.5 ppmto ca. 5.5 ppm.
 116. The method of claim 92, wherein the structuralcharacteristic is a GlcNAc or sialic acid residue and the step ofidentifying comprises determining whether the sample produces a ¹Hsignal corresponding to an acetyl methyl nucleus of a GlcNAc or sialicacid residue.
 117. The method of claim 92, wherein the structuralcharacteristic is a sialic acid residue and the step of identifyingcomprises determining whether the sample produces a ¹H signalcorresponding to an axial or equatorial H3 nucleus of a sialic acidresidue.
 118. The method of claim 92, wherein the structuralcharacteristic is a sialic acid residue with an α2-3 linkage and thestep of identifying comprises determining whether the sample produces a¹H signal corresponding to an axial H3 nucleus of a sialic acid residue.119. The method of claim 92, wherein the structural characteristic is asialic acid residue with an α2-6 linkage and the step of identifyingcomprises determining whether the sample produces a ¹H signalcorresponding to an axial H3 nucleus of a sialic acid residue.
 120. Themethod of claim 92, wherein the structural characteristic is di- ortri-acetylated NeuAc and the step of identifying comprises determiningwhether the sample produces a ¹H signal with a chemical shift at ca.2.15 ppm.
 121. The method of claim 92, wherein the structuralcharacteristic is a fucose residue and the step of identifying comprisesdetermining whether the sample produces a ¹H signal corresponding to amethyl nucleus of a fucose residue.
 122. The method of claim 92, whereinthe structural characteristic is a sialic acid and the step ofidentifying comprises determining whether the sample produces a ¹H-¹Hscalar correlation between the H3 axial and H3 equatorial nuclei of asialic acid.
 123. The method of claim 92, wherein the structuralcharacteristic is a mono-antennary Man4 residue and the step ofidentifying comprises determining whether the sample produces a ¹H-¹Hscalar correlation between the H2 and H3 nuclei of a Man4 residue. 124.The method of claim 92, wherein the structural characteristic is abi-antennary Man4 residue and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹H scalar correlationbetween the H2 and H3 nuclei of a Man4 residue.
 125. The method of claim92, wherein the structural characteristic is a galactose residue in alactosamine extension and the step of identifying comprises determiningthe chemical shifts of a ¹H-¹H scalar correlation between the H1 nucleusand another nucleus of a galactose residue.
 126. The method of claim 92,wherein the structural characteristic is a sulfated GlcNac residue andthe step of identifying comprises determining the chemical shifts of a¹H-¹H scalar correlation between the H6 nucleus and another nucleus of aGlcNac residue.
 127. The method of claim 92, wherein the structuralcharacteristic is a phosphorylated mannose residue and the step ofidentifying comprises determining the chemical shifts of a ¹H-¹H scalarcorrelation between the H6 nucleus and another nucleus of a mannoseresidue.
 128. The method of claim 92, wherein the structuralcharacteristic is a core fucose residue and the step of identifyingcomprises determining the chemical shifts of a ¹H-¹³C scalar correlationfor the anomeric nucleus of a GlcNAc2 residue.
 129. The method of claim92, wherein the structural characteristic is a sialic acid residue andthe step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto an axial H3 nucleus of a sialic acid residue.
 130. The method ofclaim 92, wherein the structural characteristic is a sialic acid residueand the step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto an equatorial H3 nucleus of a sialic acid residue.
 131. The method ofclaim 92, wherein the structural characteristic is an acetylated sialicacid residue and the step of identifying comprises determining the 1Hchemical shift of a ¹H-¹³C scalar correlation of the H7, H8 and/or H9nuclei of a sialic acid residue.
 132. The method of claim 92, whereinthe structural characteristic is a Man4 residue and the step ofidentifying comprises determining the chemical shifts of a ¹H-¹³C scalarcorrelation corresponding to an anomeric nucleus of a Man4 residue. 133.The method of claim 92, wherein the structural characteristic is a mono-or bi-antennary Man4 residue and the step of determining comprisesdetermining whether the Man4 residue is mono-antennary or bi-antennarybased on the chemical shifts of the ¹H-¹³C scalar correlation.
 134. Themethod of claim 92, wherein the structural characteristic is a Man4′residue and the step of identifying comprises determining whether thesample produces a ¹H-¹³C scalar correlation with chemical shiftscorresponding to an anomeric nucleus of a Man4′ residue.
 135. The methodof claim 92, wherein the structural characteristic is a mono- orbi-antennary Man4′ residue and the step of determining comprisesdetermining whether the Man4′ residue is mono-antennary or bi-antennarybased on the chemical shifts of the ¹H-¹³C scalar correlation.
 136. Themethod of claim 92, wherein the structural characteristic is a GlcNAc1residue and the step of identifying comprises determining whether thesample produces a ¹H-¹³C scalar correlation with chemical shiftscorresponding to an anomeric nucleus of a GlcNAc1 residue.
 137. Themethod of claim 92, wherein the structural characteristic is a GlcNAc2residue and the step of identifying comprises determining whether thesample produces a ¹H-¹³C scalar correlation with chemical shiftscorresponding to an anomeric nucleus of a GlcNAc2 residue.
 138. Themethod of claim 92, wherein the structural characteristic is a Man3residue and the step of identifying comprises determining whether thesample produces a ¹H-¹³C scalar correlation with chemical shiftscorresponding to an anomeric nucleus of a Man3 residue.
 139. The methodof claim 92, wherein the structural characteristic is a GlcNac residuewith a β(1-2) linkage to mannose and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to an anomeric nucleus of a GlcNac residuewith a β(1-2) linkage to mannose.
 140. The method of claim 92, whereinthe structural characteristic is a GlcNAc residue with a β(1-4) orβ(1-6) linkage to mannose and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to an anomeric nucleus of a GlcNAc residuewith a β(1-4) or β(1-6) linkage to mannose.
 141. The method of claim 92,wherein the structural characteristic is a GlcNAc residue in alactosamine extension and the step of identifying comprises determiningwhether the sample produces a ¹H-¹³C scalar correlation with chemicalshifts corresponding to an anomeric nucleus of a GlcNac residue in alactosamine extension.
 142. The method of claim 92, wherein thestructural characteristic is an unsubstituted galactose residue and thestep of identifying comprises determining whether the sample produces a¹H-¹³C scalar correlation with chemical shifts corresponding to ananomeric nucleus of an unsubstituted galactose residue.
 143. The methodof claim 92, wherein the structural characteristic is a galactoseresidue with an α(2-3) sialic acid attached and the step of identifyingcomprises determining whether the sample produces a ¹H-¹³C scalarcorrelation with chemical shifts corresponding to an anomeric nucleus ofa galactose residue with an α(2-3) sialic acid attached.
 144. The methodof claim 92, wherein the structural characteristic is a galactoseresidue with an α(2-6) sialic acid attached and the step of identifyingcomprises determining whether the sample produces a ¹H-¹³C scalarcorrelation with chemical shifts corresponding to an anomeric nucleus ofa galactose residue with an α(2-6) sialic acid attached.
 145. The methodof claim 92, wherein the structural characteristic is a galactoseresidue in a lactosamine extension and the step of identifying comprisesdetermining whether the sample produces a ¹H-¹³C scalar correlation withchemical shifts corresponding to an anomeric nucleus of a galactoseresidue in a lactosamine extension.
 146. The method of claim 92, whereinthe structural characteristic is an oligomannose structure and the stepof identifying comprises determining whether the sample produces a¹H-¹³C scalar correlation with chemical shifts corresponding to ananomeric nucleus of an oligomannose structure.
 147. The method of claim92, wherein the structural characteristic is a core fucose residue andthe step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto an anomeric nucleus of a core fucose residue.
 148. The method ofclaim 92, wherein the structural characteristic is a core fucose residueand the step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto a methyl nucleus of a core fucose residue.
 149. The method of claim92, wherein the structural characteristic is an antennary fucose residueand the step of identifying comprises determining whether the sampleproduces a ¹H-¹³C scalar correlation with chemical shifts correspondingto a methyl nucleus of an antennary fucose residue.
 150. The method ofclaim 92, wherein the structural characteristic is a label attached toGlcNAc1 and the step of identifying comprises determining whether thesample produces a ¹H signal corresponding to GlcNAc1αH1.
 151. The methodof claim 92, wherein the structural characteristic is a label attachedto GlcNAc1 and the step of identifying comprises determining whether thesample produces a split ¹H-¹H scalar correlation corresponding to fucoseH1/H2, H1/H3 or —CH3/H5 nuclei.
 152. The method of claim 92, wherein thestructural characteristic is a label attached to GlcNAc1 and the step ofidentifying comprises determining whether the sample produces a ¹H-¹³Cscalar correlation corresponding to an anomeric nucleus of GlcNAc1α orGlcNAc1β.
 153. The method of claim 92, wherein the structuralcharacteristic is a label attached to GlcNAc1 and the step ofidentifying comprises determining the chemical shifts of a ¹H-¹³C scalarcorrelation corresponding to an anomeric nucleus of a GlcNAc2 or Man3residue.
 154. The method of claim 92, wherein the structuralcharacteristic is a label attached to GlcNAc1 and the step ofidentifying comprises determining whether the sample produces a split¹H-¹³C scalar correlation corresponding to an anomeric fucose nucleus.155. The method of claim 92, wherein the structural characteristic is alabel attached to GlcNAc1 and the step of identifying comprisesdetermining whether the sample produces a split ¹H-¹³C scalarcorrelation corresponding to a methyl fucose nucleus.